The Human Blood Fluke, Schistosoma mansoni, Harbors Bacteria Throughout the Parasite's Life Cycle.

While symbiotic relationships between invertebrates and bacteria have been extensively described, studies of microbial communities inhabiting parasitic worms remain scarce. Exploring the microbiota associated with helminths responsible for major infectious diseases will inform on parasite biology, host-pathogen interactions, and disease pathophysiology. We investigated the presence of microorganisms inhabiting tissues of the human parasite Schistosoma mansoni. In situ hybridization using a pan-bacterial 16S rRNA gene probe revealed bacteria colonizing key developmental stages that were successfully removed after antibiotic treatment of live parasites. Understanding the composition and function of the S. mansoni-associated microbiota may lead to the development of novel microbiome-targeting control strategies.

Strongyloidiasis in humans and dogs in Southern Italy: an observational study.

Strongyloidiasis is a clinical issue both in humans and in dogs. Moreover, there are concerns about its zoonotic potential. We aimed to explore Strongyloides stercoralis epidemiology in Southern Italy in humans and dogs sharing the same environment in three different settings: (1) kennels (group K); (2) livestock farms (group L) and (3) agricultural farms (group A). For humans, a commercial ELISA test was used for screening. RT-PCR on faecal samples was done for people testing positive or equivocal at serology. On dog's faecal samples, Baermann test and RT-PCR were performed. A total of 145 dogs and 139 persons were tested. Based on faecal tests in dogs and serology in humans, a S. stercoralis positivity of 4.1% and 6.5% was revealed, respectively. The sites where cases were found were different for animals and humans. In dogs the highest positivity was in group K (6.7% against 2% and 0% in L and A). Differently, in humans the proportion of positive results was similar between the groups (p = 0.883). Fifty percent (3/6) of positive dogs were healthy; the other dogs presented weight loss and/or diarrhoea. ELISA-positive persons (n=9) were all in health, but abdominal pain (37.5%), urticaria (22.2%) and asthma (22.2%) were reported, resolving after treatment with oral ivermectin 200 µg/kg. RT-PCR performed on 13 human faecal samples resulted negative. These findings suggest that strongyloidiasis is present in humans and dogs in Southern Italy, and screening in larger cohorts would be needed for more accurate estimates.

A retrospective study comparing agar plate culture, indirect immunofluorescence and real-time PCR for the diagnosis of Strongyloides stercoralis infection.

Strongyloides stercoralis is a parasite that can cause death in immunocompromised people. A proper diagnosis is hence essential. The real-time polymerase-chain reaction (RT-PCR) is a novel, promising diagnostic method, that detects the DNA of the parasite in stool samples. In this retrospective study, we compared the sensitivity of agar plate coproculture (APC), an in-house immunofluorescence test (IFAT) and an in-house RT-PCR for the diagnosis of S. stercoralis infection. The study sample was composed by 223 samples. Samples resulting positive to APC, IFAT and RT-PCR were 20, 140 and 25, respectively. When sensitivity was calculated against a composite reference standard, serology confirmed the best performance (sensitivity 95%), followed by RT-PCR (57%) and APC (45%). In conclusion, in a non-endemic setting, serology is the best screening method, while the combination of APC and RT-PCR does not seem a reasonable approach to increase sensitivity. Both methods can have a role as confirmatory tests for selected cases.

OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation.

2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) is predicted to be a conserved 2OG oxygenase, the catalytic domain of which is related to hypoxia-inducible factor prolyl hydroxylases. OGFOD1 homologs in yeast are implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol response genes (Ofd1, Schizosaccharomyces pombe) to translation termination/mRNA polyadenylation (Tpa1p, Saccharomyces cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyzes the posttranslational hydroxylation of a highly conserved residue (Pro-62) in the small ribosomal protein S23 (RPS23). Unusually OGFOD1 retained a high affinity for, and forms a stable complex with, the hydroxylated RPS23 substrate. Knockdown or inactivation of OGFOD1 caused a cell type-dependent induction of stress granules, translational arrest, and growth impairment in a manner complemented by wild-type but not inactive OGFOD1. The work identifies a human prolyl hydroxylase with a role in translational regulation.

Serological and molecular tests for the diagnosis of Strongyloides stercoralis infection in dogs.

Strongyloides stercoralis can cause severe infection both in humans and dogs. Coproparasitological examination has low sensitivity for the diagnosis of this parasite; hence, different diagnostic techniques have been implemented. However, serology and molecular methods have been assessed almost exclusively in humans. In this study, two serologic assays and a real-time PCR (RT-PCR), routinely used for the diagnosis of strongyloidiasis in humans, have been tested for the diagnosis in dogs. Five dogs living in the same kennel in Bari, southern Italy, were diagnosed with S. stercoralis infection by detection of larvae in fecal samples processed by the Baermann method. Serum, fecal, and tissue (lungs, scraping of intestinal tract) samples from the same dogs were tested with two serologic assays (commercial ELISA, in-house IFAT) and with an in-house RT-PCR, routinely used for diagnosis in humans. IFAT was positive in all serum samples, ELISA in 3/7 (42.8%) samples. RT-PCR was positive in all pre-treatment fecal samples, in all fecal debris, and in intestinal scraping (three samples from the same deceased dog). The results suggest that IFAT and RT-PCR techniques routinely used for S. stercoralis diagnosis in humans could be useful for the diagnosis of the infection in dogs.

Characteristics of Respiratory Syncytial Virus Infections in Children in the Post-COVID Seasons: A Northern Italy Hospital Experience.

BACKGROUND: Public health measures for COVID-19 mitigation influenced the circulation of Respiratory Syncytial Virus (RSV) during the 2020-2021 winter season. In the following autumn, an unprecedented resurgence of RSV occurred. Our study monitored RSV pediatric infections one and two years after the relaxation of containment measures for the COVID-19 pandemic. METHODS: We analyzed diagnostic molecular data for SARS-CoV-2, flu, and RSV infections and clinical data from children with respiratory symptoms referring to our hospital during the 2021-2022 and 2022-2023 seasons. RESULTS: In the 2021-2022 season, the number of RSV-affected children was very high, especially for babies <1 year. The outbreak appeared in a shorter interval of time, with a high clinical severity. In the 2022-23 season, a reduced number of infected pediatric patients were detected, with a similar hospitalization rate (46% vs. 40%), and RSV accounted for 12% of the infections. Coinfections were observed in age <2 years. In RSV patients, symptoms were similar across the two seasons. CONCLUSIONS: The clinical presentation of RSV in the two post-COVID seasons suggests that the pathophysiology of the virus did not change across these two years. Further studies are needed to continuously monitor RSV to support an effective prevention strategy.

Wide Real-Life Data Support Reduced Sensitivity of Antigen Tests for Omicron SARS-CoV-2 Infections.

With the continuous spread of new SARS-CoV-2 variants of concern (VOCs), the monitoring of diagnostic test performances is mandatory. We evaluated the changes in antigen diagnostic tests' (ADTs) accuracy along the Delta to Omicron VOCs transition, exploring the N protein mutations possibly affecting ADT sensitivity and assessing the best sampling site for the diagnosis of Omicron infections. In total, 5175 subjects were enrolled from 1 October 2021 to 15 July 2022. The inclusion criteria were SARS-CoV-2 ADT combined with a same-day RT-PCR swab test. For the sampling site analysis, 61 patients were prospectively recruited during the Omicron period for nasal and oral swab analyses by RT-PCR. Next-Generation Sequencing data were obtained to evaluate the different sublineages. Using RT-PCR as a reference, 387 subjects resulted in becoming infected and the overall sensitivity of the ADT decreased from 63% in the Delta period to 33% in the Omicron period. This decrease was highly statistically significant (p < 0.001), and no decrease in viral load was detected at the RNA level. The nasal site presented a significantly higher viral load than the oral site during the Omicron wave. The reduced detection rate of Omicron infections by ADT should be considered in the global testing strategy to preserve accurate diagnoses across the changing SARS-CoV-2 variants.

Prevalence of Strongyloides stercoralis and other helminths in four districts of Madagascar.

BACKGROUND: Estimation of prevalence of Strongyloides stercoralis infection is required in endemic areas, in order to identify areas in need of control programmes. Data on prevalence of strongyloidiasis in Madagascar are scant. Aim of this work was to estimate prevalence of S. stercoralis in four districts of Madagascar. METHODS: Fecal and serum samples collected in the context of a previous study on schistosomiasis were tested with S. stercoralis real-time PCR and serology, respectively. A multiplex real-time PCR for Ascaris lumbricoides, Ancylostoma duodenalis, Necator americanus, and Trichuris trichiura was done on fecal samples collected in the areas demonstrating higher prevalence of strongyloidiasis. Comparisons between proportions were made using Fisher exact test, with false discovery rate correction used for post-hoc comparisons. A multivariable Firth logistic regression model was used to assess potential risk factors for S. stercoralis infection. RESULTS: Overall, 1775 serum samples were tested, of which 102 of 487 (20.9%) and 104 of 296 (35.2%) were serological-positive in Marovoay and in Vatomandry districts (both coastal areas), respectively, compared to 28 of 496 (5.6%) and 30 of 496 (6.1%) in Tsiroanomandidy and in Ambositra districts (both highlands), respectively (adj. p < 0.001). PCR for S. stercoralis was positive in 15 of 210 (7.1%) and in 11 of 296 (3.7%) samples from Marovoay from Vatomandry, respectively, while was negative for all samples tested in the other two districts. High prevalence of A. lumbricoides (45.9%), hookworm (44.6%) and T. trichiura (32.1%) was found in Vatomandry. In the multivariable analysis, strongyloidiasis was associated with hookworm infection. Hookworm infection was also associated with male sex and lower education level. CONCLUSIONS: S. stercoralis prevalence proved higher in coastal areas compared to highlands. Different climatic conditions may explain this distribution, along with previous rounds of anthelminthics distributed in the country, which may have reduced the parasite load in the population. The high prevalence of the other soil-transmitted helminths (STH) in Vatomandry was unexpected, given the good coverage with benzimidazole in control campaigns. Further studies are needed to explore the risk factors for STH and S. stercoralis infections in Madagascar, in order to align with the WHO recommendations.

Resurgence of Respiratory Virus after Relaxation of COVID-19 Containment Measures: A Real-World Data Study from a Regional Hospital of Italy.

The SARS-CoV-2 virus spread in the Northern Hemisphere during the 2019/2020 influenza seasons and it persisted in the 2021/2022 season. A cocirculation of SARS-CoV-2 and influenza viruses was expected in Italy during the winter seasons. This study aims to investigate the prevalence of influenza and respiratory syncytial viruses observed in a hospital in Verona Province, Italy hospital during these past three winter seasons and to compare our data with national and global surveillance reports on the transmission of respiratory viruses in the preceding decade. Our findings clearly demonstrated the extremely low prevalence of influenza virus among hospitalized patients and outpatients during the first two COVID-19 winter seasons, with a reemergence of respiratory syncytial virus in the late 2021. Containment measures may have played an important role in temporarily stopping the circulation of respiratory viruses, but after relaxation, in 2021, we experienced an unusual increase of respiratory syncytial viruses at the beginning of the winter season.

Technical Note: RNA Extraction Alternative Method for SARS-CoV-2 Molecular Diagnosis.

The devastating COVID-19 outbreak posed serious challenges for the diagnostics laboratories, facing global shortage of reagents and equipment. This study aimed at evaluating an additional RNA extraction method respect to those already recommended by WHO and CDC. A new protocol for RNA extraction from nasopharyngeal swab was set up, adapting a Qiagen kit, and validated on a set of 96 clinical samples. The analysis showed a sensitivity of 94% and a specificity of 97%, but considering samples with Ct<36.5, the sensitivity and the specificity increased to 100%. The adapted method was also able to detect samples with very low viral load (Ct>38), indicating that the two approaches can be considered equivalent for the SARS-CoV-2 diagnostics. This extraction method can help in increasing the throughput for SARS-CoV-2 molecular test, even in a low automation setting.

Helminth Microbiota Profiling Using Bacterial 16S rRNA Gene Amplicon Sequencing: From Sampling to Sequence Data Mining.

Symbiont microbial communities play important roles in animal biology and are thus considered integral components of metazoan organisms, including parasitic worms (helminths). Nevertheless, the study of helminth microbiomes has thus far been largely overlooked, and symbiotic relationships between helminths and their microbiomes have been only investigated in selected parasitic worms. Over the past decade, advances in next-generation sequencing technologies, coupled with their increased affordability, have spurred investigations of helminth-associated microbial communities aiming at enhancing current understanding of their fundamental biology and physiology, as well as of host-microbe interactions. Using the blood fluke Schistosoma mansoni as a key example of parasitic worms with complex life cycles involving multiple hosts, in this chapter we (1) provide an overview of protocols for sample collection and (2) outline an example workflow to characterize worm-associated microbial communities using high-throughput sequencing technologies and bioinformatics analyses of large-scale sequence data.

Molecular techniques for the genomic viral RNA detection of West Nile, Dengue, Zika and Chikungunya arboviruses: a narrative review.

Introduction: Molecular technology has played an important role in arboviruses diagnostics. PCR-based methods stand out in terms of sensitivity, specificity, cost, robustness, and accessibility, and especially the isothermal amplification (IA) method is ideal for field-adaptable diagnostics in resource-limited settings (RLS).Areas covered: In this review, we provide an overview of the various molecular methods for West Nile, Zika, Dengue and Chikungunya. We summarize literature works reporting the assessment and use of in house and commercial assays. We describe limitations and challenges in the usage of methods and opportunities for novel approaches such as NNext-GenerationSequencing (NGS).Expert opinion: The rapidity and accuracy of differential diagnosis is essential for a successful clinical management, particularly in co-circulation area of arboviruses. Several commercial diagnostic molecular assays are available, but many are not affordable by RLS and not usable as Point-of-care/Point-of-need (POC/PON) such as RReal-TimeRT-PCR, Array-based methods and NGS. In contrast, the IA-based system fits better for POC/PON but it is still not ideal for the multiplexing detection system. Improvement in the characterization and validation of current molecular assays is needed to optimize their translation to the point of care.

Overall decrease in SARS-CoV-2 viral load and reduction in clinical burden: the experience of a hospital in northern Italy.

OBJECTIVES: In Italy the burden of patients with coronavirus disease 2019 (COVID-19) gradually decreased from March to the end of May. In this work we aimed to evaluate a possible association between the severity of clinical manifestations and viral load over time during the epidemiological transition from high-to low-transmission settings. METHODS: We reviewed the cases of COVID-19 diagnosed at the emergency room of our hospital, retrieving the proportion of patients admitted to the intensive care unit. A raw estimation of the viral load was done evaluating the Ct (cycle threshold) trend obtained from our diagnostic reverse transcriptase real-time PCR test. RESULTS: The proportion of patients requiring intensive care significantly decreased from 6.7% (19/281) in March to 1.1% (1/86) in April, and to none in May (Fisher's test p 0.0067). As for viral load, we observed a trend of Ct increasing from a median value of 24 (IQR 19-29) to 34 (IQR 29-37) between March and May, with a statistically significant difference between March and April (pairwise Wilcoxon test with stepdown Bonferroni adjustment for multiple testing, p 0.0003). CONCLUSIONS: We observed a reduction over time in the proportion of patients with COVID-19 requiring intensive care, along with decreasing median values of viral load. As the epidemiological context changes from high-to low-transmission settings, people are presumably exposed to a lower viral load which has been previously associated with less severe clinical manifestations.

Correction: A global genotyping survey of Strongyloides stercoralis and Strongyloides fuelleborni using deep amplicon sequencing.

[This corrects the article DOI: 10.1371/journal.pntd.0007609.].

Preliminary comparison between an in-house real-time PCR vs microscopy for the diagnosis of Loa loa and Mansonella perstans.

Infections with the filarial nematodes Loa loa and Mansonella perstans are among the most neglected filarial infections. L. loa is endemic in 11 countries of Central and West Africa and loiasis is estimated to affect about 20 million people. M. perstans infection is widespread in more than 30 countries of sub-Saharan Africa. Due to the difficulty in diagnosing loiasis and M. perstans mansonellosis on a clinical basis, the diagnosis of infection with L. loa and M. perstans relies on laboratory techniques. Definitive diagnosis is based on the detection, identification, and quantification of circulating microfilariae (mf) by microscopy of concentrated blood. However, this is impractical for screening purposes as it requires expert laboratory personnel, considerable blood manipulation, and is time consuming, especially for the final issue of negative result reports, which are very common in the population visited outside endemic areas. The aim of the current work is the preliminary evaluation of the performance of the in-house real-time PCR described by Ta and colleagues compared to the routine microscopic approach for the screening of filarial infections in the clinical setting outside endemic areas, using samples from patients accessing the dedicated outpatient clinics for migrants and travelers of a reference centre for tropical diseases in Northern Italy.

Accidental diagnosis of COVID-19 pneumonia after 18F FDG PET/CT: a case series.

Purpose: The aim of this case series is to illustrate possible [18F]-FDG uptake patterns associated to COVID-19. Methods: Retrospective assessment of all Fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) scans performed for any clinical / oncological reason from 1st April 2020 to 30th April 2020. Results of PCR testing for SARS-CoV-2 were retrieved for all patients with lung consolidations and/or peripheral ground glass opacities characterized by increased metabolism to evaluate any possible association with the viral infection. Results: Seven (4%) out of 172 FDG-PET scans were included. Six out of seven patients (85%) had positive RT-PCR for SARS-CoV-2, while one patient (15%) had possible (not PCR confirmed) COVID-19 pneumonia. Conclusion: Suspicious accidental COVID-19 findings in Nuclear Medicine Department need to be reported and appropriately evaluated to implement proper supportive treatment and infection control measures.

Performance Evaluation of a Commercial Real-Time PCR Assay and of an In-House Real-Time PCR for Trypanosoma cruzi DNA Detection in a Tropical Medicine Reference Center, Northern Italy.

Chagas disease, a neglected protozoal disease endemic in Latin America, is also currently considered an emerging threat in nonendemic areas because of population movements. The detection of Trypanosoma cruzi DNA is increasingly being considered as important evidence to support Chagas disease diagnoses. However, further performance evaluation of molecular assays is useful for a standardization of strategy considering the whole process in routine diagnosis, especially for the different settings such as endemic and nonendemic countries. Seventy-five samples were collected from subjects screened for Chagas disease in Italy. The DNA was isolated from blood using automated extraction. We evaluated the performance of the commercial RealCycler® CHAG kit (pmPCR) based on satellite DNA (SatDNA) and of an in-house real-time PCR (ihPCR) targeting Sat and kinetoplast (k) DNAs, using the concordance of two serology assays as a reference standard. The sensitivity of kDNA and SatDNA tests by ihPCR and SatDNA by pmPCR were 14.29% (95% confidence interval (CI) 6.38 to 26.22), 7.14% (95% CI 1.98 to 17.29), and 7.14% (95% CI 1.98 to 17.29), respectively. Specificity was 100% for all PCR assays and targets. Overall, our results suggest that the preferred approach for clinical laboratories is to combine the kDNA and SatDNA as targets in order to minimize false-negative results increasing sensitivity.

Sensitivity, Specificity and Predictive Values of Molecular and Serological Tests for COVID-19: A Longitudinal Study in Emergency Room.

BACKGROUND: We assessed the sensitivity, specificity and positive and negative predictive value (PPV and NPV) of molecular and serological tests for the diagnosis of SARS-CoV-2 infection. METHODS: A total of 346 patients were enrolled in the emergency room. We evaluated three Reverse Transcriptase-real time PCRs (RT-PCRs) including six different gene targets, five serologic rapid diagnostic tests (RDT) and one ELISA. The final classification of infected/non-infected patients was performed using Latent Class Analysis combined with clinical re-assessment of incongruous cases. RESULTS: Out of these, 24.6% of patients were classified as infected. The molecular test RQ-SARS-nCoV-2 showed the highest performance with 91.8% sensitivity, 100% specificity, 100.0% PPV and 97.4% NPV respectively. Considering the single gene targets, S and RdRp of RQ-SARS-nCoV-2 had the highest sensitivity (94.1%). The in-house RdRp presented the lowest sensitivity (62.4%). The specificity ranged from 99.2% for in-house RdRp and N2 to 95.0% for E. The PPV ranged from 97.1% of N2 to 85.4% of E and the NPV from 98.1% of S to 89.0% of in-house RdRp. All serological tests had < 50% sensitivity and low PPV and NPV. VivaDiag IgM (RDT) had 98.5% specificity, with 84.0% PPV, but 24.7% sensitivity. CONCLUSION: Molecular tests for SARS-CoV-2 infection showed excellent specificity, but significant differences in sensitivity. Serological tests have limited utility in a clinical context.

Blastocystis prevalence and subtypes in autochthonous and immigrant patients in a referral centre for parasitic infections in Italy.

In this study we characterized the presence and subtype (ST1-ST4) of Blastocystis in patients attended at a referral center for tropical diseases in Northern Italy. We also, evaluated the organism's association with other intestinal parasites. Parasite screening was performed on 756 patients, from different geographical origins (namely, Italians, Africans, South Americans, Asian and non-Italian Europeans) in which Italians represented the largest group. Blastocystis was seen to be the most prevalent parasite in the study. Subtype 3 and 1 were the most frequently found in the Italians and Africans. Our data confirmed previous studies performed in Italy, in which ST3 proved to be the most prevalent subtype, but we highlighted also a high frequency of mixed subtypes, which were probably underestimated in former analyses. Interestingly, the mixed subtypes group was the most prevalent in all the analysed geographical areas. About half of our cases showed other co-infecting parasites and the most frequent was Dientamoeba fragilis. Our study confirms that, in Blastocystis infection, multiple subtypes and co-infecting parasites are very frequently present, in particular Dientamoeba fragilis.

Use of Real-Time Polymerase Chain Reaction to Differentiate between Pathogenic Entamoeba histolytica and the Nonpathogenic Entamoeba dispar in Ecuador.

Microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of intestinal parasites. Recently, polymerase chain reaction (PCR) has been approved by the World Health Organization as the method of choice for the diagnosis of Entamoeba histolytica. Of the 106 stool samples collected from the Esmeraldas and Pichincha provinces of Ecuador, all (100%) were positive for E. histolytica/Entamoeba dispar by light microscopy, whereas using real-time PCR (RT-PCR) DNA amplification, 74 (69.8%) were positive for E. dispar and only three (2.8%) were positive for E. histolytica. Some 29 (27.4%) samples were negative for the presence of either E. histolytica or E. dispar, this may be due the presence of Entamoeba mosksvskii, which is morphologically identical to E. histolytica/E. dispar and not specifically targeted by the RT-PCR used. These results indicate the necessity of reevaluating the epidemiology of amebiasis in Ecuador as the prominent species found are nonpathogenic.